This application is a Divisional Application of U.S. Ser. No. 11/445,475, filed May 31, 2006, now U.S. Pat. No. 8,163,881, which claims benefit of priority to U.S. Provisional Application Ser. No. 60/685,934, filed May 31, 2005, the entire content of which are hereby incorporated by reference.
1. Field of the Invention
The present invention relates generally to the fields of immunology and molecular biology. More specifically, the invention relates to modified Ig molecules with improved characteristics.
2. Description of the Related Art
Immunoglobulin G (IgG) constitutes the most prevalent immunoglobin class in the serum of man and other mammals. Despite fluctuations in rates of synthesis by B cells, IgGs are maintained at remarkably constant levels in the serum. If IgG homesostasis is disturbed, then pathology due to too high (hypergammaglobunemia) or too low (hypogammaglobunemia) can result. Studies indicate that the major histocompatibility complex (MHC)-class I related receptor, FcRn, is involved in the homeostasis of serum IgGs (Ghetie et al., 1996; Junghans and Anderson, 1996; Israel et al., 1996). This receptor most likely acts as a salvage receptor, and this would be consistent with its known ability to transcytose IgGs in intact form across the neonatal gut (Wallace and Rees, 1980; Rodewald and Kraehenbuhl, 1984) and yolk sac (Roberts et al., 1990; Israel et al., 1995) or placenta (Kristoffersen and Matre, 1996; Simister et al., 1996; Leach et al., 1996; Firan et al., 2001). More recent studies indicate that FcRn is also involved in the transport of IgGs across epithelial and endothelial cell barriers of diverse origin (Antohe et al., 2001; McCarthy et al., 2000; Spiekermann et al., 2002; Dickinson et al., 1999; Kobayashi et al., 2002; Yoshida et al., 2004), and this has relevance to the delivery of IgG to different sites in the body. Thus, the use of protein engineering to modify the interaction site of an IgG with FcRn offers a way of modulating the serum persistence, distribution and transport of that antibody.
The interaction sites of FcRn on mouse IgG1 (mIgG1) and human IgG1 (hIgG1) have been mapped using site-directed mutagenesis of recombinant Fc-hinge fragments, followed by analysis of these fragments both in vivo and in vitro (Kim et al., 1994b; Medesan et al., 1996; Medesan et al., 1997; Kim et al., 1999). From these studies, 1253 (EU numbering (Edelman et al., 1969)), H310, H435 and to a lesser extent, H436 (Y436 in hIgG1) play a central role in this interaction. These amino acids are located at the CH2-CH3 domain interface (Deisenhofer, 1981), and the mapping of the functional site to these residues is consistent with the crystallographic structure of rat FcRn complexed with rat Fc (Burmeister et al., 1994b; Martin et al., 2001).
The FcRn interaction site encompasses three spatially close loops comprised of sequences that are distal in the primary amino acid sequence. The central role of Fc histidines in building this site accounts for the marked pH-dependence (binding at pH 6.0, release at pH 7.2-7.4) of the Fc-FcRn interaction (Rodewald and Kraehenbuhl, 1984; Raghavan et al., 1995; Popov et al., 1996), as the pKa of one of the imidazole protons lies in this pH range. This pH dependence is essential for the release of FcRn bound IgG molecules when they come to the cell surface following intracellular recycling or transcytosis (Ghetie and Ward, 2000; Ober et al., 2004a). I253, H310, H435 and to a lesser degree, H436, are highly conserved in IgGs of both human and rodent IgGs (Kabat et al., 1991). This, taken together with the isolation of a human homolog of FcRn (Story et al., 1994), indicate that the molecular mechanisms involved in IgG homeostasis and transport are common to both mouse and man and this has implications for the modulation of pharmacokinetics, distribution and delivery of IgGs to different body sites.
In studies to identify the FcRn interaction site on Fc, mutations of Fc fragments (comprising the Fc and hinge region) have been made that reduce the serum half-lives of the corresponding Fc fragments (Medesan et al., 1997; Kim et al., 1994a; Kim et al., 1999). In addition, Fc fragments or IgGs with increased affinity for binding to FcRn have been engineered (Ghetie et al., 1997; Shields et al., 2001; Hinton et al., 2004) and these molecules have increased serum persistence in mice (Ghetie et al., 1997) or cynomologous monkeys (Hinton et al., 2004).
Immunoglobulin Fc domains are also of great interest for purposes of studying the mechanisms of antibody interactions with further molecules of the immune system. These include, depending on the class of antibody, interactions with complement, and binding to specific receptors on other cells, including macrophages, neutrophils and mast cells. More detailed knowledge of the biology of Fc regions is important in understanding various molecular processes of the immune system, such as phagocytosis, antibody-dependent cell-mediated cytotoxicity and allergic reactions.
The production of a longer-lived Fc fragment or antibody having increased binding to Fc receptors is attractive, since such a fragment or antibody can be used, for example, to tag therapeutic reagents. This allows fewer doses of the agent to be used in therapy and possibly even allows lower doses of the agent to be used through its increased persistence in the bloodstream. Additionally, such molecules would be useful, in and of themselves, for therapy against pathogenic agents, cancer and autoimmune diseases. Such antibodies would also be predicted to be more efficiently transported across the placenta during the third trimester of pregnancy when FcRn is active in the maternal fetal transport of IgGs (Simister, 2003). As such, protective antibodies (e.g., anti-pathogen) could be delivered to the developing fetus.
In addition, there are multiple situations in which increased clearance of IgGs from the circulation would be desirable, e.g., in autoimmune diseases such as systemic lupus erythematosus where circulating autoreactive antibodies cause pathology, and in situations where toxins or drugs are to be cleared rapidly from the body using an antibody as a clearing agent. Increased clearance of an antibody should be achievable by using a molecule, such as an engineered antibody, that binds to FcRn with high affinity and does not dissociate rapidly at near neutral pH (unlike naturally-occurring antibodies). Such antibodies would not be released from cells, but would instead be predicted to remain bound to FcRn and block binding of other, lower affinity IgGs. As a result, FcRn function would be blocked and endogenous or therapeutic IgGs would be directed into the lysosomal pathway for degradation (Ober et al., 2004b). The targeting of such ‘blocking’ antibodies to FcRn might also be useful in the prevention of transport of pathogenic (e.g., autoreactive) antibodies from mother to fetus during pregnancy.